Wnt signaling is known for its roles in regulation of embryonic development and in tumorigenesis. The first Wnt gene was identified by virtue of its ability to induce mammary tumors in mice. Studies have now implicated Wnt signaling in many different types of tumors. The best characterized Wnt signaling pathway, the canonical Wnt signaling pathway, is initiated by the binding of canonical Wnts to their receptor complexes consisting of the LDL receptor-related protein (LRP) 5/6 and frizzled (Fz) proteins, which eventually leads to the stabilization of -catenin and activation of gene transcription. Despite remarkable advances in our understanding of Wnt signaling mechanisms, significant gaps still remain. These include the mechanism by which Wnt regulates LRP phosphorylation, an early intracellular event and the biochemical basis for the involvement of Wnt co-receptor Fz and downstream signaling molecule Dishevelled (Dvl) in the Wnt/2-catenin signaling pathway. By screening a siRNA library for human kinases, we identified a group of phosphatidylinositide (PtdIns) kinase siRNAs that could inhibit canonical Wnt signaling. We went on demonstrating that Wnt3a could induce the accumulation of PtdIns (4,5) P2 in mammalian cells, which depends on both Fz and Dvl. We also showed that PtdIns (4,5) P2 is required for Wnt3a-induced canonical signaling events. Moreover, we found that there is possible involvement of heterotrimeric G proteins in Wnt-regulated PtdIns (4,5) P2 formation. Furthermore, we have preliminary results to support the two possible mechanisms by which PtdIns (4,5)P2 may regulate LRP phosphorylation. Putting all of these together, we hypothesize that Wnt3a may, via Fz, Dvl and G, activate PtdIns kinases to stimulate the formation of PtdIns (4,5)P2, which in turn stimulates the phosphorylation of LRP5/6, an early Wnt signaling event. Specially, in this application we will: 1) Investigate the mechanisms by which Wnt3a induces the formation of PtdIns (4,5)P2. 2) Investigate the mechanisms by which PtdIns (4,5)P2 regulates LRP phosphorylation. PUBLIC HEALTH RELEVANCE: Cell-cell communication is essential for normal functions of multi-cellular organisms including human. Problems in this process would lead to various pathological conditions including tumorigenesis. This research proposal is to understand how the Wnt protein, one of the key molecules in cell-cell communication, regulate cellular functions and how the defects in this mechanism would cause diseases.